Despite the great advances in antiviral therapy for human immunodeficiency virus (HIV) infection, a successful global intervention for prevention and treatment of HIV infection will require an effective vaccine. Since the induction of cytotoxic T-lymphocyte (CTL) responses is now believed to be an important component in an effective HIV-1 vaccine, our approach to develop an HIV vaccine is based on the stimulation of CD8+ CTL response more efficiently than the natural infection. This will be accomplished by using mixture-based synthetic combinatorial libraries (SCLs) to identify optimized peptide ligands that would be effective as immunogens in stimulating T-cell mediated immune responses against HIV infection. The mixture-based SCL approach allows the rapid identification of highly active compounds from large pools of individual compounds. In particular, when generated in a positional scanning (PS) format, the key amino acid(s) of the active peptide sequence(s) can be determined directly from the initial screening of the library. CTLs recognize processed viral peptides generally 8 to 11 amino acids in length, which are presented as a molecular complex with MHC class I and Beta2-microglobulin. PS-SCLs of nonapeptides and decapeptides (having a carboxylic acid or carboxamide C-terminus, and an acetylated or non-acetylated N-terminal amino group) will therefore be used for the proposed studies. Each mixture will be screened for their ability to stimulate cytokine production and/or cytolytic activity by CTL clones having specificity for immunodominant epitopes of the Gag, reverse transcriptase, and Nef proteins. Following the deconvolution processes to identify epitope mimics from the libraries, the CTL reactivities to the identified agonists will be determined, and their immunogenicity will be assessed by performing in vitro stimulation experiments. Thus, the immunologic reactivity of cells pulsed with peptides to the immunizing native peptide and/or identified mimics will be measured by cytokine release or chromium release assays. Furthermore, the cross-recognition of the most potent identified peptides will be investigated using a cohort of HLA-A2-positive patients. Although not yet applied to HIV research, the success of the PS-SCL approach has been reported for the identification of peptides being several order of magnitude more effective than native peptide ligands to stimulate clonotypic populations of autoreactive CD4+, and tumor-specific or alloreactive CD8+ T-cells.